The membrane mucin Muc4 is overexpressed in a variety of carcinomas. Some evidence has accumulated suggesting that Muc4 may have roles in both the initial transformation of healthy epithelia to early hyperplastic phenotypes and in latr transitions to highly invasive and metastatic states. For example, it has been reported that Muc4 is overexpressed and mislocalized in 20% of local breast tumors compared to patient-matched normal tissue samples. Coupled with observations that Muc4 expression is sufficient to transform mouse fibroblasts, these observations raise the possibility that Muc4 plays an active role in initiating events in breast cancer. It has also been reported that Muc4 protein is highly upregulated in the majority of breast cancer lymph node metastases compared to patient-matched primary tumors, pointing to a role in later events related to progression to metastasis. The studies outlined here seek to rigorously assess the roles of Muc4 in breast cancer onset and progression using the MCF10A progression series, an isogenic series of cell lines modeling normal, hyperplastic, and malignant breast tissue. The hypothesis driving the proposed studies is that Muc4 overexpression engages specific signaling pathways to promote cellular transitions to progressively more malignant states. The study will be broken down into two specific aims. Specific Aim 1 will characterize the impact of Muc4 overexpression on the in vitro growth properties of the MCF10A progression series cell lines. In these experiments, the MCF10A progression series cells will be stably transduced with tetracycline-inducible Muc4, and the impact of Muc4 on cellular transformation, proliferation, motility, and invasiveness will be measured, along with Muc4-dependent potentiation of growth and survival signaling pathways. These studies will also examine the impact of Muc4 overexpression on the growth of cells in three dimensional cell cultures using the Matrigel matrix. Specific Aim 2 will determine whether Muc4 overexpression induces transitions from less malignant to more malignant states in the normal mammary environment in vivo. The cell lines created in Aim 1 will be orthotopically introduced into nude mouse mammary fat pads, and tumor latency, size, and rate of metastasis will be measured without and with Muc4 induction. Histological analysis of collected tissues will be employed to assess Muc4-induced transitions in cancer stage. The successful completion of the proposed studies could implicate Muc4 as a novel target for future therapeutic intervention in breast cancer. Training opportunities for the PI include classes in mouse biology, pathology and surgery, weekly meetings with the UCD mammary gland biology and pathology group, an extensive series of seminars, tumor boards and symposia in cancer biology and therapeutics, and close collaboration with pathologists and statisticians. Results emerging from the study will be presented at a national conference in cancer biology in Year 3. PUBLIC HEALTH RELEVANCE: The role of the mucin Muc4 signaling in breast cancer formation and progression. The proposed studies are aimed at understanding the molecular and cellular mechanisms underlying the initiation of breast cancer and the progression of breast cancer lesions to malignancy. The successful completion of the work could shed new light on the biochemical and cellular mechanisms engaged by tumor cells to facilitate their transformation and transition to a malignant state. Ultimately, the studies could identify a novel target for therapeutic intervention.